Flow cytometryによる睾丸内精子形成能の評価判定 第1報: ヒト睾丸組織内DNA量分布測定の基礎的検討

Abstract

ヒト睾丸組織内DNA量分布をflow cytometryを用い測定するための試料作成法を検討した.1)ヒト睾丸組織は, 0.05%collagenase中で37°C 60分間処理で, 遊離細胞にすることができた.2)遊離細胞を固定しないで処理するKrishan法は, ヒト睾丸組織の試料作成法としては不適当であった.3)遊離細胞を70%エタノール固定後, RNase, pepsin処理し, PIで蛍光染色, flow cytometryを用いDNA量を測定すると, 良好なヒストグラムを得た.4) Pepsin処理でcell aggregation, cell debrisは著明に減少し, ヒストグラムが良好となったが, RNase処理はヒストグラムに特徴的変化を示さなかった.5) DNAヒストグラムを解析して, 睾丸組織内のspermを除いたhaploid, diploid, tetraploidの細胞比率を短時間に, 正確に測定できた.以上より, ヒト睾丸組織を適切な試料作成法を用い処理し, flow cytometryでDNA量分布を測定すると, 精子形成能の定量的評価となりうる可能性を示唆した

The present study was carried out to establish the best method of preparing human testicular tissue for flow cytometric DNA analysis including dispersal, fixation and staining. Human testicular tissue could be dispersed to single cells by incubating in 0.05% collagenase solution at 37 degrees C for 60 minutes. Krishan's method which stains nuclear DNA directly without ethanol fixation and digestion in ribonuclease was not suitable for testicular cells. After ethanol fixation, testicular cells were treated with ribonuclease and pepsin, then stained with propidium iodide. Nuclear DNA in cells was measured by flow cytometry and a good DNA histogram was obtained. Ribonuclease influenced the DNA histogram little, but pepsin markedly improved it by digesting cell debris and decreasing cell aggregation. Analysis of the DNA histogram revealed the proportion of haploid, diploid and tetraploid cells accurately and quickly. Flow cytometric DNA analysis could be a useful method of evaluating cell kinetics in spermatogenesis.